1. Field of the Invention
This invention relates to a method for preparing antigens comprising a peptide-protein complex and to a method for preparing antibodies using the antigens.
2. Description of the Prior Art
An antigen is a substance which stimulates a living organism to produce a specific antibody and react therewith specifically. That is, an antigen is a substance which has an antigenicity capable of forming an antibody in vivo and an immunoreactivity capable of reacting with the antibody in vitro. Representative examples of antigens are foreign proteins, such as bacteria, viruses, various toxins, etc. On the other hand, an antibody is substance produced in a living organism, e.g., mammals, etc., due to the stimulation of an antigen and is present mainly in blood sera, in particular, in the .gamma.-globulin fraction in blood sera. Antibodies can react with antigens in vitro or in vivo.
Antigens have on the surface thereof one or more determinant groups which can react with antibodies and antibodies have on the surface thereof one or more reacting groups which can combine with the determinant group(s). These two substances have a complementary spatial structure to each other called by Ehrlich a "key and key hole relationship" to which the specificity between antigens and antibodies is ascribable.
Most of the naturally found antigens are considered to have on the surface thereof a mosaic of various determinant groups and specific antibodies are considered to be produced corresponding to the respective determinants.
The antigen-antibody reaction occurring between antigens having common determinant groups and the corresponding antibodies is termed a "cross-reaction" or "group reaction".
An antigen-antibody reaction comprises a first step in which the antigen combines with a corresponding antibody in a very short time (e.g., 20 seconds) even at low temperatures, e.g., 0.degree. C. to form a conjugater, and a second step in which the conjugates thus obtained combine with each other to cause agglutination which can be observed visually.
One of the characteristics of an antigen-antibody reaction is high specificity and such reaction has widely been utilized in the diagnosis of various diseases.
The determination of hormones has conventionally been conducted using an immunoreaction, i.e., an antigen-antibody reaction, as is well known in the art.
Pancreatic glucagon is one of the physiological pancreatic hormones which plays an important role in the uptake and metabolism of sugars and has the following amino acid sequence ##STR2##
Quantitative determination of pancreatic glucagon has recently attracted much attention in the fields of diagnosis, pathology and the like since the determination of the level of pancreatic glucagon in blood makes it possible to diagnose various diseases or pathological states, for example, diabetes, etc.
The conventional method for determining pancreatic glucagon has been performed using a radioimmunoassay method (hereinafter referred to as RIA method) in which antibodies obtained using pancreatic glucagon per se or antigens which contain pancreatic glucagon as a hapten are employed. This RIA method is successful to some extent. [see Unger et al: Proc. Soc. Exp. Biol. Med. 102, 621 (1959)].
However, as research in the art proceeds the presence of glucagon-like substances which behave immunologically in a manner similar to pancreatic glucagon has been discovered in the digestive tract or gut of mammals and such is named "gut-GLI". [see Sutherland et al: J. Biol. Chem. 175, 663, (1964) and Unger et al: Metabolism, 15, 865 (1966)] and it has also been discovered that the antibodies obtained using pancreatic glucagon per se or antigens which contain pancreatic glucagon as a hapten cross react not only with pancreatic glucagon but also with gut-GLI's so that they are non-specific to pancreatic glucagon. In other words in determining pancreatic glucagon using the RIA method, the above-described known antibodies give values which include values ascribable to the reaction with gut-GLI's resulting in a failure to provide a diagnostically exact determination.
It has recently been reported that pancreatic glucagon-specific antibodies were obtained by chance using the same antigen as described above [see Diabetes, 17 (Suppl. 1), 321 (1968)].
However, it is also reported that these antibodies which are called "G 58" and "30 K" can be obtained only by chance and very rarely [see Saishin Igaku (Newest Medicine) 30 (11) p 1901 (1975) and Heading, L.G.: Horm. Metab. Res., 1, 87-88 (1969)]. Therefore, the method for preparing antibodies using an antigen which contains pancreatic glucagon as a hapten has poor reproducibility and is not practically acceptable.
On the other hand, the structure-function relationship of glucagon has been investigated [see Assan et al: Diabetes, 21, 843, (1972)] by synthesizing various peptides related to glucagon, i.e., C.sub.1 to C.sub.23 fragments, and determining their biological activity using the increase in blood sugar level as an indicator. Further, investigations were made on antigenic sites in the glucagon molecule using a glucagon-specific antibody "K 47" and a glucagon non-specific antibody "PVP 8" leading to the assumption that there are at least two antigenic sites in the glucagon molecule and that one of these antigenic sites is present in the C-terminal portion, in particular the C.sub.23 -C.sub.29 fragments, of the glucagon molecule.
Instead of progress in the investigation of the mechanism of the immunological behavior of glucagon or gut-GLI as above, however, no reports on the development of practically acceptable antibodies have been made.
As stated above, known antibodies are either non-specific to pancreatic glucagon or can be obtained with poor reproducibility so that development of techniques which can be used to produce antibodies with specificity to pancreatic glucagon on an industrial scale is strongly desired.